1. Field of the Invention
This invention relates to a method for reproducibly generating hepatitis A virus (HAV) in high (milligram) yields. Specifically, this invention relates to a process for cytopathically generating a hepatitis A virus in a cell line.
2. Description of the Background Art
An propagated in persistently-infected cells has traditionally been used to generate antigen for immunodiagnostic purposes such as HAVAB kits produced by Abbott Laboratories in North Chicago, Ill. A single milligram of HAV using their system has been estimated as costing $25,000 (K. Watson, 1984), and involving a single person full-time for a year for virus purification.
Since the first demonstration of cell culture growth of hepatitis A virus (HAV) by Provost and Hilleman (1979), there have been numerous reports describing conditions for growth of HAV in various cell lines and under various conditions (Flehmig, 1980; 1981; Flehmig et al., 1981; Daemer et al., 1981; Gauss-muller et al., 1984; Bradley et al., 1984; Vallbracht et al., 1984; Simmons et al., 1985; Wheeler et al., 1986a). After initial adaptation of the virus to the appropriate cell line, the growth of hepatitis A virus in cell culture has been reported as being non-cytopathic and usually results in a persistent infection in which both cells and virus coexist in a symbiotic relationship. Quantitation of virus present within persistently infected cells determined by cell culture infectivity or physical particle counts has varied from 10.sup.5 virus particles/ml up to 10.sup.10 virus particles/ml (Binn et al., 1984; Simmons et al., 1985; Wheeler et al., 1986a, b). Recently there have been reports of HAV in cell culture producing degeneration of host cells (Venuitti et al., 1985; Shen et al., 1986; Anderson et al., 1986; 1987; Cromeans et al., 1987). The conditions necessary to produce the cytopathic effect in each system appear to be cell and growth condition specific and the quantity of purified virus obtained has not been reported.
A human fecal sample obtained during the acute phase of hepatitis A infection and propagated in FRhK4 cells was designated HAS-15 (Bradley et al., 1984). The quantity of virus produced, as evaluated by radioimmunoassay, appeared to increase with passage, while the time required for viral growth decreased. Subsequently, the ninth passage of this strain was passaged more than 20 times at 7-day intervals to select for a rapidly growing virus population (Wheeler et al., 1986a). Using large scale virus propagation methods, the purified virus derived from these persistently-infected cells was used to characterize the HAV capsid polypeptides (Wheeler et al., 1986b). The initial yield from 350 liters of cell supernatant and the cell lysates from 1.times.10.sup.6 cm.sup.2 of cells was approximately 5 mg of purified virus (Wheeler et al., 1986b), although subsequent recoveries were substantially lower.
The adaptation of HAV for growth in cell culture from fecal samples has been shown to require an initial protracted lag or eclipse phase (Frosner et al., 1979; Daemer et al., 1981; Gauss-Muller et al., 1981; Binn et al., 1984; and Bradley et al., 1984) varying from 4 weeks to 12 weeks. After adaptation, which can be dependent upon the type of cells and/or the titer of the original inocula, passage of virus-positive cell lysates or supernatants, results in a decrease in the time needed for detectable viral growth and an apparent increase in the amount of virus produced. However, in none of these reports has there been any evidence of cytotoxicity associated with HAV replication.
In contrast to the early extended time periods needed for adaptation of virus from clinical specimens, cell-culture adapted virus becomes detectable by immunoassay or immunofluorescence within 1-17 days (Vallbracht et al., 1985; Simmonds et al., 1985; Wheeler et al., 1986a; and Cromeans et al., 1987) and this relatively rapid growth has been utilized to grow HAV by passage of persistently-infected cells (Vallbracht et al., 1984; Simmonds et al., 1985; and Wheeler et al., 1986b). However, although persistent infection has been purported to be the most practical method to grow HAV, the data indicates that over a period of time there was a significant decrease in the amount of virus produced by persistently infected cells. The cause of this decrease was not clear. One explanation was that over a long period of time, cells more sensitive to HAV growth were destroyed by infection, and a population of cells resistant to HAV infection was selected. This would explain the decreased virus production and the observation that the majority of the virus was cell-associated.
A previous report from the inventors' laboratory indicated that persistent infection of FRhK4 cells can produce large quantities of virus (Wheeler et al., 1986b), however, the time course data and ongoing experience suggests that this method does not sustain high-yield production of virus and that the major portion of the virus was probably obtained from the early phase of the persistent infection as well as from the acute infection used to initiate the persistently infected cells.
There are now four examples of cytopathic infection with HAV isolates which have been reported (Venutti et al., 1985; Shen et al., 1986; Anderson et al., 1986, 1987; and Cromeans et al., 1987), two of which are derived from separate lines of persistently-grown HM-175. It has been suggested that, in the case of one of the HM-175 cytopathic variants (Anderson et al., 1987), that the cytopathic nature and mechanism of persistence is associated with an alteration in the capsid polypeptide composition. Although this possibility cannot be ruled out, nucleotide sequence analysis of the long-term persistently grown HAS-15, which causes cell degeneration when used for an acute infection at high multiplicity of infection revealed that within the first 700 nucleotides of the VP1 molecule or the most surface exposed and potentially variable capsid polypeptide within other picornaviruses, there was only a single nucleotide difference when compared to the nucleotide sequence of lower passage (20 times) HAS-15 (Ovchinnikov et al., 1985).
The industry is lacking a process for reliably and economically reproducing milligram quantities of hepatitis A virus from cell culture propagation.